Studies have shown that discharged Coronavirus disease 2019 (COVID-19) patients have retested positive for SARS-CoV-2 during a follow-up RT-PCR test. We sought to assess the results of continued nucleic acid testing for SARS-CoV-2 patients in COVID-19 patients after they were discharged in Lu’an, China.We conducted RT-PCR tests on sputum, throat swabs, fecal or anal swabs, and urine samples collected from 67 COVID-19 patients following discharge. Samples were collected on the 7th and 14th days following discharge. Patients testing positive on the 7th or 14th day were retested after 24 hours until they tested negative twice.Seventeen  discharged COVID-19 patients had a positive RT-PCR retest for SARS-CoV-2. Among them, 14 (82.4%) were sputum positive, five (29.4%) were throat swab positive, seven (41.2%) were fecal or anal swab positive, one (5.9%) was urine sample positive, five (29.4%) were both sputum and throat swab positive, four (23.5%) were both sputum and fecal test positive, and one (5.9%) was positive of all four specimens. The shortest period of time between discharge and the last positive test was 7 days, the longest was 48 days, and the median was 16 days. The proportion of positive fecal or anal swab tests increased from the third week. The median Cq cut-off values after onset were 26.7 after the first week, 37.7 the second to sixth week, and 40 after the sixth week. There were no significant differences between the RT-PCR retest positive group and the unrecovered positive group.There was a high proportion of patients who retested positive for COVID-19. Discharge criteria have remained fairly consistent so we encourage regions affected by COVID-19 to appropriately amend their current criteria. There hCOVID-19 patients discharged in China were RT-PCR tested and found to be positive for SARS-CoV-2 again during follow-up care . Other chttps://www.who.int/news-room/commentaries/detail/criteria-for-releasing-covid-19-patients-from-isolation;https://www.who.int/publications/i/item/clinical-management-of-covid-19). Countries have also updated their discharge protocols. For example, according to the new Korea Centers for Disease Control (KCDC) discharge protocols, “no additional tests are required for cases that have been discharged from isolation”  has updated its criteria for discharging isolated COVID-19 patients; in this criteria retesting is not required  normal temperature (<37.3 °C) for 3 days; 2) reduced respiratory tract symptoms and clear clinical improvement confirmed by pulmonary imaging, and 3) collection of two consecutive negative RT-PCR tests on respiratory samples at least 24 h apart .An epidemiological investigation was conducted and all 67 patients were confirmed to have maintained distance from others and remained indoors after discharge, thus ruling out possible reinfection. Sputum, throat, fecal or anal swabs, and urine samples were collected on the 7th and 14th day following discharge. If a patient tested negative, a second test was performed more than 24 h after the first test until the patient was confirmed to be negative for two consecutive tests. The patient was immediately required to continue the quarantine protocol in the hospital for at least 14 days.RT-PCR was used to conduct nucleic acid testing. The RT-PCR kit was manufactured by Sun Yat-sen University Da’an Gene Co., Ltd., China (20203400063). The ROC curve was used to determine the ORF1 ab and N reference values, which according to the test results of the clinical samples resulted in Cq values equal to 40. Samples were determined to be negative when amplification above 40 was detected. Samples with amplification below cycle 37 indicated that the diagnosis was positive. Testing was repeated for samples with a Cq range of 37–40. A repeated Cq value less than 40 with an amplification curve that had obvious fluctuations indicated a positive result; otherwise the result was determined to be negative.A retested positive case was defined as one that was positive for SARS-CoV-2 in any specimen when RT-PCR tested following discharge. Recovered patients who were considered negative were those who remained negative from discharge to the end of the study period. Secondary cases were the cases resulting from a secondary transmission.p < 0.01 was considered to be statistically significant.Data were double-entered using EpiData software, version 3.1  and were analyzed using SPSS software version 11.0 . Pearson Chi-square was used to analyze and count data in different groups. The T test was used to compare the mean between groups. Tests were designed as two-tailed and an alpha value of The research protocols used in this study were reviewed and approved by the Lu’an Municipal Health and Family Planning Commission and written informed consent was obtained from all research participants.Seventeen  discharged COVID-19 patients retested positive for SARS-CoV-2 using RT-PCR testing. Fourteen (82.4%) were sputum positive, five (29.4%) were throat swab positive, seven (41.2%) were fecal or anal swab positive, one (5.9%) was urine sample positive, five (29.4%) were both sputum and throat swab positive, four (23.5%) were both sputum and fecal swab positive, and one (5.9%) was positive in all four specimens .Among the 17 cases, 11 (64.7%) tested positive more than once following discharge and six (35.3%) tested positive only once. The shortest amount of time between discharge and the last positive test was 7 days, the longest was 48 days, and the median interval was 16 days .p = 0.226) were observed between the average number of days required for the continued collection of positive fecal or anal swab samples (19.0 ± 10.2 days) and other specimens (14.2 ± 5.5 days) following discharge, whereas the proportion of positive fecal or anal swab samples increased from the third week . Our hope is that we can increase the preventive measures and control the infection of this virus in China and other areas by introducing revised discharge criteria.The seventh iteration of the COVID-19 diagnosis and treatment plan in China emphasizes the health management and isolation of patients following discharge. We identified criteria between positive patients who recovered and those who did not recover in order to aid the early screening process. However, no significant differences were noted among common epidemiological characteristics, the clinical characteristics, and the disease status, which is inconsistent with the findings of other relevant studies. Our study has some deficiencies and limitations. Firstly, due to the limitations of on-site investigations, we mainly used descriptive epidemiological investigation methods and, as a result, could not explore the specific mechanism for recovering positivity in discharged patients. Secondly, due to the limited sample size, we did not find a difference in the characteristics of recovered positive and unrecovered positive patients. Therefore, a larger cohort is needed to verify our conclusions. Thirdly, virus culture tests need to be performed in laboratories that reach biosafety level 3. As we did not have access to such resources, this will be our focus in future studies.We confirm that there is a high probability of testing positive after COVID-19 patients are discharged. China’s discharge criteria have remained fairly consistent so we encourage vulnerable regions to appropriately strengthen discharge criteria for COVID-19 patients, despite not being able to verify their infectivity. We hope that there will be more opportunities to conduct viral cell culture tests along with RT-PCR in biosafety level 3 laboratory conditions to better understand and verify the infectivity of COVID-19. This will help us better develop and improve the discharge criteria.10.7717/peerj.11617/supp-1Supplemental Information 1The results of the analysis in the article.Click here for additional data file.


P = 0.04), but not significantly influenced by clinicopathological features. However, miR-143-3p expression in colonic tumors was rare for upregulation, although there was a significant difference by existence of desmoid tumors. MiR-143-3p transfection significantly inhibited colorectal cancer cell proliferation compared to control microRNA transfection. Our data suggested regulation of miR-143-3p expression differed by samples (plasma or colonic tumors) in most FAP patients. Upregulation of plasma miR-143-3p expression may be helpful for diagnosis of FAP, although suppressive effect on tumorigenesis seemed insufficient in FAP patients.Circulating microRNAs (miRNAs) are considered promising biomarkers for diagnosis, prognosis, and treatment efficacy of diseases. However, usefulness of circulating miRNAs as biomarkers for hereditary gastrointestinal diseases have not been confirmed yet. We explored circulating miRNAs specific for patients with familial adenomatous polyposis (FAP) as a representative hereditary gastrointestinal disease. Next-generation sequencing (NGS) indicated that plasma miR-143-3p, miR-183-5p, and miR-885-5p were candidate biomarkers for five FAP patients compared to three healthy donors due to moderate copy number and significant difference. MiR-16-5p was considered as an internal control due to minimum difference in expression across FAP patients and healthy donors. Validation studies by real-time PCR showed that mean ratios of maximum expression and minimum expression were 2.2 for miR-143-3p/miR-16-5p, 3.4 for miR-143-3p/miR-103a-3p, 5.1 for miR-183-5p/miR-16-5p, and 4.9 for miR-885-5p/miR-16-5p by using the samples collected at different time points of eight FAP patients. MiR-143-3p/16-5p was further assessed using specimens from 16 FAP patients and 7 healthy donors. MiR-143-3p was upregulated in FAP patients compared to healthy donors ( MicroRNAs (miRNAs) are non-coding RNAs that regulate multiple biological processes in diseases through the post-transcriptional regulation of gene expression, including in cancer, cardiovascular disease, and neurodegenerative disease , 2. CircAPC) tumor suppressor gene, which results in hundreds to thousands of colorectal adenomatous polyps Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: NoReviewer #2: Partly**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: NoReviewer #2: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: “Assessment of circulating microRNA specific for patients with familial adenomatous polyposis” by Yamano and colleagues is an effort to downselect a cohort of microRNAs from a group of clinical samples obtained from 15 familial adenomatous polyposis and 7 controls. Using next generation sequencing the authors identify miRs that may be potential biomarkers for pathology.While the study is strengthened by the observation of clinical findings, the lack of any functional studies diminishes the enthusiasm in the study and the potential of the findings. In order for the study to be improved the following sets of data are highly encouraged to be introduced.1. Gain and loss of functional stides usingselected microRNAs. Judicious choice of controls are warranted to strengthen the observations2. What are the targets being regulated by the microRNAs of interests and how are they affecting the disease process?3. Experimental evidence to highlight the biological/ physiological importance of occurrence of miRs in biosamples such as plasma and tumors- and also if there is an occurrence at 1 site, do they lose the potential to be biomarkers?Reviewer #2: In this manuscript by Yamano et al, the authors are looking for circulating miRNA in patients with a hereditary disease called familial adenomatous polyposis. Both plasma and tissue miRNA expression was tested from patients with FAP vs control (tumor or mucosa for tissue).1. The authors provide no rationale for performing the experiments. The expression of APC is used for FAP, and given that this is a hereditary disease, there is often family history present as well. The authors make no argument to the need of another marker for FAP. They describe in the introduction that they are going to compare 3 groups: healthy vs. FAP vs. CRC, and that potentially they could find a marker that would span both FAP and non-hereditary CRC. This wasn't done.2. It's unclear why the authors use two control miRNAs for expression of miR143-3p, but not for any of the other potential candidates.3. The Figures are mislabeled. There are only 2 figures (with A & B on both).4. The expression of miRNA in the tumors is mostly decreased compared to control tissue, but there is no significant change. No explanation is provided for the difference between plasma and tumor tissue.Overall, the major concern with this manuscript is the lack of rationale for the studies and the lack of description of the results.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  30 Aug 2020Dear Reviewers,Thank you for your time for reviewing the manuscript and describing the useful comments to us. We have revised the manuscript and responded to your comments as possible as we could.We apologized to the reviewers for not performing the appropriate basic experiments to support our findings, although we agree the importance of the further experiments. We considered that new samples from the FAP patients or APC deficient mice were most suitable for the experiments. However, performing the experiments is beyond our capacity now. Then, we added some data to support our findings and mainly revised Abstract and Discussion to clearly describe the findings and problems of our study.The association between plasma miR-143-3p and pathological T factor / lymph node metastasis and the rarity of mir-143-3p upregulation in colonic tumors were added in the revised manuscript.Discussion was organized by circulating miR-143-3p, tumor miR-143-3p, inconsistent expression between plasma and tumor, target of mir-143-3p, speculation, and limitation of the study, because these contents were described roughly in the first version. Reviewer #1: “Assessment of circulating microRNA specific for patients with familial adenomatous polyposis” by Yamano and colleagues is an effort to downselect a cohort of microRNAs from a group of clinical samples obtained from 15 familial adenomatous polyposis and 7 controls. Using next generation sequencing the authors identify miRs that may be potential biomarkers for pathology.While the study is strengthened by the observation of clinical findings, the lack of any functional studies diminishes the enthusiasm in the study and the potential of the findings. In order for the study to be improved the following sets of data are highly encouraged to be introduced.1. Gain and loss of functional studies using selected microRNAs. Judicious choice of controls are warranted to strengthen the observations.Thank you for your comment. Many studies using cancer cell lines have already confirmed onco-suppressive effect of mir-143-3p. Our data showed that circulating mir-143 was upregulated in FAP patients, although it seemed no significant influence on tumorigenicity by upregulated miR-143-3p. Then, we considered that basic studies using colonic samples from FAP patients or APC deficient mice may be more suitable for functional study than cancer cell lines. However, these experiments were beyond our capacity now. Instead of functional studies, we added the data regarding association between plasma mir-143-3p and pathological data  in Results. We also assessed tendency of miR-143-3p expression in colonic tumors and described in Results. We also revised Discussion.ResultsValidation(First version) Line: 176-178There was no significant difference in miR-143-3p expression based on patient sex (P = 0.29), levels before or after radical surgery (P = 0.43), the existence of desmoid tumor (P = 0.33), or family history (P =0.91).(Revised version) Line200-204There was no significant difference in miR-143-3p expression based on pathological T factor , pathological node positive or not (P =0.42), patient sex (P = 0.29), levels before or after radical surgery (P = 0.43), the existence of desmoid tumor (P = 0.33), or family history (P =0.91).MiR-143-3p expression in colonic tumors(First version) No description.(Revised version) Line:216-217Then, upregulation was significantly rare by chi-square test (P =0.02).Discussion(First version) Line215-217Our study had several limitations. First, our results regarding the upregulation of circulating miR-143 and downregulation of colonic tumor miR-143 has not been confirmed in studies using APC-deficient mice.(Revised version) Line: 257-260Our study had several limitations mainly because we have described the data from clinical samples, which were not supported by basic experiments. First, biological effect of miR-143-3p have not been evaluated by using colonic samples derived from FAP patients or APC-deficient mice. 2. What are the targets being regulated by the microRNAs of interests and how are they affecting the disease process?Thank you for your comment. Target genes of mir-143-3p against cancer cell lines have been reported already as we described in Discussion. In this study, upregulated mir-143-3p in FAP patients seemed ineffective to prevent tumorigenesis. Then, the specimens from FAP patients or APC deficient mice may be more preferable than cancer cell lines to assess the target genes. However, these experiments were beyond our capacity.The source cells of upregulated miR-143-3p expression are also critical to explain our findings. We have added the sentence in revised Discussion as below.Discussion(First version) Line194-196There are many target genes of the tumor suppressor function of miR-143 in CRC, including Bcl-2, CASP3, CD44, KLF5, KRAS, BRAF, TLR2, SOD1, IGF1R, ITGA6, and ASAP3 [14-19].(Revised version) Line:249-252There are many target genes of the tumor suppressor function of miR-143 in CRC, including Bcl-2, CASP3, CD44, KLF5, KRAS, BRAF, TLR2, SOD1, IGF1R, ITGA6, and ASAP3 [27-32]. However, these results were performed using the cancer cell lines, but not benign tissues or cells from FAP patients or APC deficient mice. 3. Experimental evidence to highlight the biological/ physiological importance of occurrence of miRs in biosamples such as plasma and tumors- and also if there is an occurrence at 1 site, do they lose the potential to be biomarkers?The reason why mir-143-3p showed different expression pattern by the location was undetermined. The critical issue should be the cellular source of plasma mir-143-3p, which is also elevated in the patients with cerebral atherosclerosis, acute ischemic stroke, and amyotrophic lateral sclerosis. Mesenchymal cells such as fibroblasts and smooth muscle cells have been reported to highly express mir-143-3p compared to colonic epithelial cells. Well-organized experiments are necessary for understanding the mechanisms of mir-143-3p in human.We added the sentences in Discussion as below.Discussion (First version) Line:196-197However, our current results suggested the upregulation of plasma miR-143-3p was insufficient for inhibiting tumor progression in patients with FAP.(Revised version) Line:245-248Then, our current results were inconsistent with the previous studies because upregulation of plasma miR-143-3p seemed insufficient for inhibiting tumor progression in FAP patients. Difference of miR-143-3p expression by sample location and its interaction has not been evaluated. (First version) Line:212-214Our finding that suggests that the upregulation of mir-143-3p in colonic tumor is associated with desmoid tumor should be validated by further studies. Desmoid tumor is considered a serious neoplasm related with FAP and difficult to diagnose [32].(Revised version) Line: 261-264Second, the sources of upregulated plasma miR-143-3p have not been confirmed. Possible sources of mir-143 could be mesenchymal cells such as fibroblast and smooth muscle cells [34]. Then, higher expression of miR-143-3p in colonic tumor with desmoid tumor (fibromatosis) in our study seemed to be associated with this speculation. [35]. Reviewer #2: In this manuscript by Yamano et al, the authors are looking for circulating miRNA in patients with a hereditary disease called familial adenomatous polyposis. Both plasma and tissue miRNA expression was tested from patients with FAP vs control (tumor or mucosa for tissue).1. The authors provide no rationale for performing the experiments. The expression of APC is used for FAP, and given that this is a hereditary disease, there is often family history present as well. The authors make no argument to the need of another marker for FAP. They describe in the introduction that they are going to compare 3 groups: healthy vs. FAP vs. CRC, and that potentially they could find a marker that would span both FAP and non-hereditary CRC. This wasn't done.We hypothesized that microRNA profiling in genetic diseases should be modified to compensate the disadvantages induced by genetic abnormality. Then, we considered that FAP patients should have the specific profiling of circulating microRNAs to compensate the influence by loss of APC function. We selected FAP as a representative genetic disease among gastrointestinal diseases, because we have constantly experienced FAP patients and published manuscripts for several decades. Another representative genetic disease, Lynch syndrome, has variable clinical and genetic features than FAP.Most FAP patients are usually diagnosed by clinical features such as colonic polyposis and family history without confirming APC mutation. Diagnosis of Lynch syndrome is more difficult than that of FAP , and CRC patients are not routinely assessed for Lynch syndrome. We expected that effective diagnosis by circulating microRNAs for FAP could be applied for Lynch. We apologize that description is so insufficient that you misunderstand the study. The description showed our hypothesis that circulating microRNAs profiling maybe specific for FAP patients, which should be different from healthy patients and sporadic CRC patients. We have not assessed blood samples derived from sporadic CRC patients.We have revised the sentences as below. And added reference 10 regarding FAP and reference 13 regarding Lynch syndrome.Abstract (First version) Line: 34-36We assessed circulating miRNAs of patients with familial adenomatous polyposis (FAP) as a hereditary disease and prototype of colorectal cancer progression.(Revised version) Line:28-31We assessed circulating miRNAs of patients with familial adenomatous polyposis (FAP) as a hereditary disease and prototype of colorectal cancer progression to evaluate as possible biomarkers of hereditary gastrointestinal diseases.Introduction(First version) No description(Revised version) Line: 64-65We have experienced hundreds of FAP patients for several decades [10].(First version) Line:77-80Therefore, we chose FAP as a representative hereditary disease and a prototype of CRC and attempted to identify and assess a plasma miRNA specific for patients with FAP as a potential biomarker.(Revised version) Line:71-77Although most FAP cases are diagnosed by endoscopic examination without further genetic test, application of endoscopic examination for younger family is usually difficult to perform. Lynch syndrome, another hereditary disease, is difficult for diagnosis by endoscopic examination alone, and needs further examinations including genetic test [13]. Therefore, we chose FAP as a representative hereditary disease to evaluate the possibility of circulating miRNA as a potential biomarker of hereditary gastrointestinal diseases.Discussion(First version) Line:189-191In the current study, we explored circulating miRNAs specific for patients with FAP and found that miR-143-3p was upregulated in the plasma of patients with FAP, but not in colonic tumors.(Revised version) Line:231In the current study, we explored circulating miRNAs specific for FAP patients.(First version) Line:217-218Second, we did not validate the usefulness of measuring circulating miR-143 in family members of patients with FAP.(Revised version) Line: 265-266Third, we did not validate the usefulness of measuring circulating miR-143-3p in family members of FAP patients, who were not diagnosed with FAP or not. 2. It's unclear why the authors use two control miRNAs for expression of miR143-3p, but not for any of the other potential candidates.Different from tumor, control miRNA for blood samples has not been confirmed, although miR-103-3p is considered as one of the common control miRNAs. Next generation sequence data indicated miR-16-5p was suitable control miRNA, because of similar expression between FAP patients and healthy donors. Then, we assessed these two miRNAs. Mir-103a-3p is one of the internal control miRNAs of blood samples recommended by manufacture’s instruction from Exiqon.Abstract(First version) Line:39-40With minimum differences among samples, miR-16-5p was selected as an internal control.(Revised version) Line:34-35MiR-16-5p was also assessed as an internal control due to minimum difference in expression across FAP patients and healthy donors. ResultsExploration of potential plasma biomarkers of FAP(First version)No description in Results.(Revised version) Line:172-174NGS also showed that miR-16-5p was most stably expressed across FAP patients and healthy donors, which indicated a good candidate for normalizer in quantitative PCR study. 3. The Figures are mislabeled. There are only 2 figures (with A & B on both).Thank you for your comment. We revised the figure numbers as below.ResultsExploration of potential plasma biomarkers of FAP(First version) Line:158Fig.1(Revised version) Line:166Fig.1AAssessment of miRNA expression fluctuations(First version) Line:169Fig. 2(Revised version) Line:166Fig.1BValidation(First version) Line: 175Fig. 3(Revised version) Line:200Fig 2A (First version) No description(Revised version) Line:202Fig 2BMiR-143-3p expression in colonic tumors(First version) Line: 181Fig. 2B(Revised version) Line:219Fig 34. The expression of miRNA in the tumors is mostly decreased compared to control tissue, but there is no significant change. No explanation is provided for the difference between plasma and tumor tissue.We statistically evaluated mir-143-3p expression in colonic tumors.Material and methodsStatistical analysis(First version) No description(Revised version) Line:160-161The proportion of category was assessed by chi-square test.ResultsMiR-143-3p expression in colonic tumors(First version) Line: 180-182The expression of miR-143-3p in the colonic tumors was upregulated with fold change more than two in one patient . Nine patients exhibited less than half expression in normal mucosa compared to the matched tumor samples.(Revised version) Line:214-217The expression of miR-143-3p in the colonic tumors was upregulated with fold change more than two in one patient alone (Table 3). Nine patients exhibited less than half expression in normal mucosa compared to the matched tumor samples. Then, upregulation was significantly rare by chi-square test (P =0.02).Discussion(First version) Line: 189-191In the current study, we explored circulating miRNAs specific for patients with FAP and found that miR-143-3p was upregulated in the plasma of patients with FAP, but not in colonic tumors.(Revised version) Line:231-232We found that miR-143-3p was upregulated in plasma, but not in colonic tumors. (First version) Line: 196-197However, our current results suggested the upregulation of plasma miR-143-3p was insufficient for inhibiting tumor progression in patients with FAP.(Revised version) Line:243-248Rarity of miR-143-3p upregulation in colonic tumors of FAP patients is consistent with a previous report [26].Then, our current results were inconsistent with the previous studies because upregulation of plasma miR-143-3p seemed insufficient for inhibiting tumor progression in FAP patients. Difference of miR-143-3p expression by sample location and its interaction has not been evaluated.Overall, the major concern with this manuscript is the lack of rationale for the studies and the lack of description of the results.Thank you for your comment. We revised introduction and discussion and added the data.Our rationale is described in Introduction as below.IntroductionRevised version, Line:71-77Although most FAP cases are diagnosed by endoscopic examination without further genetic test, application of endoscopic examination for younger family is usually difficult to perform. Lynch syndrome, another hereditary disease, is difficult for diagnosis by endoscopic examination alone, and needs further examinations including genetic test [13]. Therefore, we chose FAP as a representative hereditary disease to evaluate the possibility of circulating miRNA as a potential biomarker of hereditary gastrointestinal diseases.Description of the results were described as below.Discussion(First version) Line: 189-191In the current study, we explored circulating miRNAs specific for patients with FAP and found that miR-143-3p was upregulated in the plasma of patients with FAP, but not in colonic tumors.(First version) Line: 203-207Circulating miRNAs are promising biomarkers for the diagnosis of CRC [21–23]. However, only a few reports have considered miR-143 as a biomarker of CRC [24–26]. On the other hand, miR-143 has been reported as a candidate biomarker for other diseases, including acute ischemic stroke, cerebral atherosclerosis, pituitary adenoma, and ulcerative colitis [27–30].(Revised version) Line231-237We found that miR-143-3p was upregulated in plasma, but not in colonic tumors. Circulating miRNAs are promising biomarkers for the diagnosis of CRC [15-17]. However, only a few reports have considered miR-143 as a biomarker of CRC [18-20]. On the other hand, circulating miR-143 has been upregulated in other diseases including acute ischemic stroke, cerebral atherosclerosis, amyotrophic lateral sclerosis, and pituitary adenoma [21-24].Reference 24 has been changed from the manuscript about ulcerative colitis to amyotrophic sclerosis, because the former manuscript described regarding colonic tumors, but not blood samples.(First version) Line:196-197However, our current results suggested the upregulation of plasma miR-143-3p was insufficient for inhibiting tumor progression in patients with FAP.(First version) Line:200-202Our finding of the downregulation of miR-143 expression in colonic tumors of patients with FAP is consistent with a previous report [20].(Revised version) Line243-248Rarity of miR-143-3p upregulation in colonic tumors of FAP patients is consistent with a previous report [26].Then, our current results were inconsistent with the previous studies because upregulation of plasma miR-143-3p seemed insufficient for inhibiting tumor progression in FAP patients. Difference of miR-143-3p expression by sample location and its interaction has not been evaluated.Dear Reviewer 1 and Reviewer 2Under reconstruction of the manuscript, we have changed the style of Abstract and remove one sample from analysis. Re-analysis has not changed significance of the data. 1. We have also revised the abstract depending on the journal style.2. We re-analyzed the data of colonic tumors, because the first version included the sample (F20) regarding SBA data, not colonic tumor. Abstract(First version)Background Circulating microRNAs (miRNAs) are considered promising biomarkers for diagnosis, prognosis, and treatment efficacy of malignancies. We assessed circulating miRNAs of patients with familial adenomatous polyposis (FAP) as a hereditary disease and prototype of colorectal cancer progression.(Revised version)Circulating microRNAs (miRNAs) are considered promising biomarkers for diagnosis, prognosis, and treatment efficacy of malignancies. We assessed circulating miRNAs of patients with familial adenomatous polyposis (FAP) as a hereditary disease and prototype of colorectal cancer progression to evaluate as possible biomarkers of hereditary gastrointestinal diseases.(First version)Methods MiRNAs specific for patients with FAP were identified using next-generation sequencing (NGS). Those that had moderate copy numbers and significant differences among the samples were selected for further evaluation as candidate biomarkers. With minimum differences among samples, miR-16-5p was selected as an internal control. The miRNAs were assessed by real-time PCR for expression ratios between minimum and maximum values at different time points. The miRNAs with the least expression variation were validated in patient plasma and colonic tumors.Results MiR-143-3p, miR-96-5p, miR-183-5p, and miR-885-5p were selected as candidate biomarkers. Mean expression ratios of miR-143-3p/miR-16-5p, miR-143-3p/miR-103-3p, miR-183-5p/miR-16-5p, and miR-885-5p/miR-16-5p were 2.2, 3.4, 5.1, and 4.9, respectively. MiR-143-3p, with lowest variation ratio, was further assessed using specimens from 16 patients with FAP and 7 healthy donors. Plasma miR-143-3p was upregulated in FAP patients compared to healthy donors (P = 0.04), but not significantly influenced by sex, radical surgery, existence of desmoid tumor, or family history. However, miR-143-3p expression was not upregulated in colonic tumors of most patients with FAP, except for those with desmoid tumors.Conclusions Our data suggested miR-143-3p expression differed between plasma and colonic tumors of most patients with FAP. Upregulation of plasma miR-143-3p expression may be helpful in the diagnosis of FAP, although it has no suppressive effect on tumor progression.(Revised version)Next-generation sequencing (NGS) indicated that plasma miR-143-3p, miR-183-5p, and miR-885-5p were candidate biomarkers for five FAP patients compared to three healthy donors due to moderate copy number and significant difference. MiR-16-5p was also assessed as an internal control due to minimum difference in expression across FAP patients and healthy donors. Then, plasma samples were further assessed by real-time PCR. Mean ratios of maximum expression and minimum expression were 2.2 for miR-143-3p/miR-16-5p, 3.4 for miR-143-3p/miR-103a-3p, 5.1 for miR-183-5p/miR-16-5p , and 4.9 for miR-885-5p/miR-16-5p by using eight FAP samples collected at different time points. Then, miR-143-3p/16-5p was further assessed using specimens from 16 FAP patients and 7 healthy donors. Plasma miR-143-3p was upregulated in FAP patients compared to healthy donors (P = 0.04), but not significantly influenced by pathological T factor, lymph node metastasis, sex, radical surgery, existence of desmoid tumor, or family history. However, miR-143-3p expression in colonic tumors was rare for upregulation, although there was a significant difference by existence of desmoid tumors. Our data suggested regulation of miR-143-3p expression differed by samples (plasma or colonic tumors) in most FAP patients. Upregulation of plasma miR-143-3p expression may be helpful for diagnosis of FAP, although suppressive effect on tumorigenesis seemed limited in FAP patients.ResultsMiR-143-3p expression in colonic tumors(First version) Line-181-186Nine patients exhibited less than half expression in normal mucosa compared to the matched tumor samples. There was no significant difference in miR-143-3p expression based on sex (P = 0.63), pathology of colonic tumor (P = 0.81), existence of gastric (P = 0.23) or duodenal polyp (P = 0.72), or family history (P = 0.36). However, there was a significant difference in miR-143-3p expression based on the existence or absence of desmoid tumor (P = 0.0002).(Revised version) Line: 215-221Eight patients exhibited less than half expression in normal mucosa compared to the matched tumor samples. Then, upregulation was significantly rare by chi-square test (P =0.02). There was no significant difference in miR-143-3p expression based on sex (P = 0.55), pathological T factor , existence of gastric (P = 0.14) or duodenal polyp (P = 0.59), or family history (P = 0.43). However, there was a significant difference in miR-143-3p expression based on the existence or absence of desmoid tumor (P <0.0001).AttachmentResponse to Reviewers.docxSubmitted filename: Click here for additional data file. 3 Nov 2020PONE-D-20-13212R1Assessment of circulating microRNA specific for patients with familial adenomatous polyposisPLOS ONEDear Dr. Yamano,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.==============================Please address the comments provided by the reviewers. Especially reviewer #1 wants to see functionality via clinical samples or in vitro experiments. Final decision will be made based on the response .==============================plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.Please submit your revised manuscript by Dec 18 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at Please include the following items when submitting your revised manuscript:A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocolsIf applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: We look forward to receiving your revised manuscript.Kind regards,Sripathi M Sureban, Ph.D.Academic EditorPLOS ONEAdditional Editor Comments (if provided):Associate Editor:Please address the comments provided by the reviewers. Especially reviewer #1 wants to see functionality via clinical samples or in vitro experiments. Final decision will be made based on the response .Reviewer #1:The authors have failed to address the comments raised about functionality. To simply cite other evidence and not generate any new results to address the queries is not enough. Also how is including "pathological T factor, lymph node metastasis" data association with miR of any help to understand miR specific disease function.If the clinical samples were not available to perform the assays asked for the authors may want to use primary cells to address the experimental design.Reviewer #2:While the authors have addressed many of the reviewer comments, the rationale for this study is still lacking. Profiling for FAP patients would be done when? Would this happen after they have polyps? If that's the case, then based on age and other parameters, APC tests might be done. Lynch syndrome and FAP have different phenotypes, both of which are different than non-genetic CRC.This has to be clarified well.[Note: HTML markup is below. Please do not edit.]Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.Reviewer #1: (No Response)Reviewer #2: (No Response)**********2. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: PartlyReviewer #2: Partly**********3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: Yes**********4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: Yes**********5. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: NoReviewer #2: No**********6. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: (No Response)Reviewer #2: While the authors have addressed many of the reviewer comments, the rationale for this study is still lacking. Profiling for FAP patients would be done when? Would this happen after they have polyps? If that's the case, then based on age and other parameters, APC tests might be done. Lynch syndrome and FAP have different phenotypes, both of which are different than non-genetic CRC.This has to be clarified well.**********what does this mean?). If published, this will include your full peer review and any attached files.7. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  16 Feb 2021Dear Reviewers,Thank you for your time for reviewing the manuscript and describing the useful comments to us again. We have revised the manuscript and responded to your comments as possible as we could.Associate Editor:Please address the comments provided by the reviewers. Especially reviewer #1 wants to see functionality via clinical samples or in vitro experiments. Final decision will be made based on the response .Thank you for giving us to revise the manuscript again. We have performed in vitro experiments depending on Reviewer 1 suggestion and also revised the manuscript depending on Reviewer 2 suggestions. Reviewer #1:The authors have failed to address the comments raised about functionality. To simply cite other evidence and not generate any new results to address the queries is not enough. Also how is including "pathological T factor, lymph node metastasis" data association with miR of any help to understand miR specific disease function.If the clinical samples were not available to perform the assays asked for the authors may want to use primary cells to address the experimental design.Thank you for your comments. We have performed functional experiments to respond your comments using colorectal cancer cell lines, Caco2, HCT116, LoVo, and RKO instead of clinical samples. In these experiments, we assessed effect of mir-143-3p transfection on proliferation of these cells together with transfection reagent alone and transfection with control microRNA. Although there are several reports regarding effect of mir-143-3p on proliferation of colorectal cancer cells, the methods and results were different among the reports. Our results were most similar with that reported by Luo et al, which was added in References No. 32).We added the sentences below in Abstract, Material and methods, Results, and Discussion.Abstract, Line 46-47MiR-143-3p transfection significantly inhibited colorectal cancer cell proliferation compared to control microRNA transfection.Introduction, Line 83-84We also assessed the effect of the candidate miRNA on proliferation of colorectal cancer cells.Material and methods, Line 165-189Cell cultureHuman CRC cell lines, Caco2, HCT116, LoVo, and RKO were kind gifts from Dr. Hirofumi Yamamoto . These cells were authenticated by ATCC service. These cells except for RKO were cultured by Roswell Park Memorial Institute 1640 medium with 10% fetal bovine serum (FBS). RKO was cultured by Dulbecco’s modified Eagle’s medium with FBS. Culture media and FBS was purchased from Nacalai Tesque  and Atlas Biologicals  , respectively. Cell experiments were maintained at 37℃ in a humidified atmosphere of 5% CO2. Cell transfection Cells were seeded in 96-well flat-bottom plate at a density of 5000 cells/well for Caco-2, 500 cells/well for HCT116, 1000 cells/well for LoVo, and 500 cells/well for RKO, respectively. 24 hours after seeding, cells were treated with Lipofectamine 2000 and Opti-MEM containing 5 pmol of miRNA Mimic Negative Control (Control treatment), 5 pmol of hsa-miR-143-3p (miR-143 treatment), and no miRNA (Mock treatment) according to the manufacturer’s instructions. Transfection reagents including miRNAs were purchased from Thermo Fisher Scientific. Cell proliferation assay In vitro growth was evaluated using alamarBlue Cell Viability Reagent at one, two, three, and four days after transfection. The ration of the fluorescence intensity at 590 nm by treatment groups compared to Mock treatment was calculated. Effect of miR-143-3p on cell growth was evaluated by comparison of the ratio between miR-143 treatment and Control treatment using Student’s t-test.Results, Line 267-283Effect of miR-143-3p on the proliferation of colorectal cancer cells　We assessed the effect of miR-143-3p on the proliferation of CRC cell lines . Except for Caco2, miR-143 treatment showed significant growth inhibition on these cell lines compared to Control treatment at four days after transfection . MiR-143 treatment also showed significant inhibition of HCT116 and LoVo compared to Control treatment at three days after transfection . In Caco2, effect of miR-143-3p on proliferation was not significant at four days after transfection .Fig. 4. The effect of miR-143-3p on the proliferation of colorectal cancer cells.The effect of miR-143-3p on the cell growth was evaluated at one, two, three, and four days after transfection. There was no significant effect on proliferation of Caco2 (A). MiR-143-3p showed significant effect on proliferation of HCT116 (B) and LoVo (C) at three and four days after transfection (p < 0.01). MiR-143-3p showed significant effect on proliferation of RKO (D) at four days after transfection (p < 0.01). Results are expressed as mean of the ratio of cell proliferation compared to transfection without miRNA ± standard deviation. * p <0.01 compared to Control treatment.Discussion, Line 296-299Our data showed that transfection of miR-143-3p inhibited proliferation of CRC cell lines, although the extent of growth inhibition differed among these cell lines. Our results are similar to the previous reports, especially by Luo et al . Discussion, Line 306-308However, transfection of miR-143-3p should be more effective on cell proliferation than plasma miR-143-3p. Discussion, Line 320-321First, biological effect of miR-143-3p have not been evaluated by using colonic samples derived from FAP patients or APC-deficient mice, although we have shown inhibitory effect of miR-143-3p on proliferation by using CRC cell lines. Reviewer #2:While the authors have addressed many of the reviewer comments, the rationale for this study is still lacking.Thank you for your comment. Our rationale is described in Introduction as below.“Circulating miRNAs are recognized as diagnostic or prognostic biomarkers of many diseases [3]; however, their usefulness in the diagnosis of gastrointestinal hereditary diseases has not been reported [4–6]. Genetic variants in patients with hereditary disease may induce consistent and specific miRNA profile differences in their blood compared to healthy donors.”“However, we hypothesized that plasma miRNA profile of FAP patients, which bears APC variants in all cells, may differ from that of patients with sporadic CRC and healthy donors.”However, these sentences could not introduce the readers what we have done in this study as your comments. We revised Abstract and added the sentences below in Introduction.Abstract(Previous version) Line 28-31Circulating microRNAs (miRNAs) are considered promising biomarkers for diagnosis, prognosis, and treatment efficacy of malignancies. We assessed circulating miRNAs of patients with familial adenomatous polyposis (FAP) as a hereditary disease and prototype of colorectal cancer progression to evaluate as possible biomarkers of hereditary gastrointestinal diseases.(Revised version) Line 30-33Circulating microRNAs (miRNAs) are considered promising biomarkers for diagnosis, prognosis, and treatment efficacy of diseases. However, usefulness of circulating miRNAs as biomarkers for hereditary gastrointestinal diseases have not been confirmed yet. We explored circulating miRNAs specific for patients with familial adenomatous polyposis (FAP) as a representative hereditary gastrointestinal disease.Introduction(Revised version) Line 81-84In this study, we explored the candidate plasma mir-RNAs specific for FAP patients by Next-generation sequencing (NGS) at first and validated the candidate miRNA by further experiments using clinical samples. We also assessed the effect of the candidate miRNA on proliferation of colorectal cancer cells. Profiling for FAP patients would be done when? Would this happen after they have polyps? If that's the case, then based on age and other parameters, APC tests might be done. Lynch syndrome and FAP have different phenotypes, both of which are different than non-genetic CRC. This has to be clarified well.Thank you for comments regarding the diagnosis of FAP. We diagnosed FAP by clinical features, not by genetic test, because cost of genetic test is not supported by insurance in Japan. Then, the patients are charged several thousand dollars for genetic test. Then, the patients usually do not request for genetic test. Endoscopic findings of stomach (gastric polyp) and duodenum  are helpful for distinguish between FAP and Lynch. Different from FAP, genetic test for Lynch is supported by insurance in Japan.In this study, 16 of 18 patients who had gastric polyps and/or duodenal polyps could be diagnosed as FAP, not Lynch, without genetic test. We have revised the sentences as below.Material and methods(Previous version) Line 83-86Ethylenediaminetetraacetic acid containing plasma samples were collected from 18 patients clinically diagnosed endoscopically with FAP. The patients included those with and without a family history of FAP and were treated at our hospital between 2015 and 2018.(Re-revised version) Line 90-95Ethylenediaminetetraacetic acid containing plasma samples were collected from 18 patients who were diagnosed as FAP by clinical features including family history and endoscopic findings in not only colon and rectum but also stomach and duodenum. Genetic tests for diagnosis of FAP have not been performed in this study, because these tests were not covered by insurance in Japan. These patients were treated at our hospital between 2015 and 2018.AttachmentResponse to Reviewers comments 2021 0214.docxSubmitted filename: Click here for additional data file. 31 Mar 2021Assessment of circulating microRNA specific for patients with familial adenomatous polyposisPONE-D-20-13212R2Dear Dr. Yamano,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Sripathi M Sureban, Ph.D.Academic EditorPLOS ONEAdditional Editor Comments :Great work on the role of miR-143 - Congratulations!Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.Reviewer #1: All comments have been addressedReviewer #2: All comments have been addressed**********2. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: (No Response)**********3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: (No Response)**********4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: (No Response)**********5. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: (No Response)**********6. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: (No Response)Reviewer #2: (No Response)**********what does this mean?). If published, this will include your full peer review and any attached files.7. 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However, in-depth chemical and structural characterization of the CNF surface chemistry is still an open challenge, especially for low weight percentage of functionalization. This currently prevents the development of efficient, cost-effective and reproducible green synthetic routes and thus the widespread development of targeted and responsive drug-delivery CNF carriers. We show in this work how we use dynamic nuclear polarization (DNP) to overcome the sensitivity limitation of conventional solid-state NMR and gain insight into the surface chemistry of drug-functionalized TEMPO-oxidized cellulose nanofibrils. The DNP enhanced-NMR data can report unambiguously on the presence of trace amounts of TEMPO moieties and depolymerized cellulosic units in the starting material, as well as coupling agents on the CNFs surface (used in the heterogeneous reaction). This enables a precise estimation of the drug loading while differentiating adsorption from covalent bonding (∼1 wt% in our case) as opposed to other analytical techniques such as elemental analysis and conductometric titration that can neither detect the presence of coupling agents, nor differentiate unambiguously between adsorption and grafting. The approach, which does not rely on the use of  DNP-enhanced solid-state NMR unravels the surface chemistry of functionalized nanocellulose. An important milestone was achieved by Turbak and co-workers in the 1980s with the introduction of a new type of cellulosic material, called cellulose nanofibrils (CNFs),3–5 which triggered a strong and lasting scientific enthusiasm. Indeed, in addition to the characteristics inherited from cellulose, such as widespread availability, biodegradability and biocompatibility, CNFs also present excellent mechanical properties, 5 to 20 nm width fibers and a high specific area (∼100 m2 g−1) that results in extended tunable surface chemistry.6 CNFs can be organized into different 2D and 3D nano-structures such as films, membranes, hydrogels or aerogels. At the end of the 2000s, a particular grade of CNF was designed by pretreating the cellulose fibers by means of a TEMPO-mediated oxidation.7,8 These TEMPO-oxidized CNFs (CNF-t) bear aldehyde and carboxylic acid groups at the nanofiber surface that offer new opportunities for functionalization strategies.Wood-derived cellulose finds industrial applications in a wide range of fields, from paper products to buildings, cosmetics, foodstuffs or medical industry,9 For example, equivalent performances to commercial products were obtained in 2016 for CNF-t gel formulations containing five times less ibuprofen, providing a proof of concept for the efficiency of drug delivery for such systems.10 In 2017, the release profile for six active pharmaceutical ingredients (APIs) incorporated in CNF-t hydrogels was also investigated, demonstrating a stable behavior towards freeze-drying and subsequent successful rehydration.11 Notably, one of the drugs studied, an antibacterial compound metronidazole was further tested in interaction with different cellulose derivatives in order to better control its release.12 At this stage it is important to note that these studies only relied so far on drug adsorption mechanisms. It is thus important to investigate the loading of drugs, such as metronidazole, onto CNF-t through covalent binding with the aim to provide a better-controlled drug release on-demand.Drug delivery is one of the promising fields of application for CNF-t in the biomedical industry.e.g. etherification, amidation, esterification, and sialylation.13–16 These approaches mostly rely so far on the use of organic solvents such as DMF/DMSO and optimized reagents such as pyridine, carbodiimides, etc. to induce grafting at the C6 position on the CNF.15 Subsequent steps typically involve click-chemistry reactions, as described by Sharpless et al. in 2001, to further attach on to the linker an additional molecule of interest. Within this context, Diels–Alder reactions, which are compatible with these principles, have been recently implemented to produce new materials, using in particular the furan–maleimide strategy.17,18 In the biomedical context, the Diels–Alder reaction has also been used to immobilize multicolor fluorescent probes onto CNF for biological imaging16 and to produce enzymatically activated oligosaccharide-prodrugs of doxorubicin.19Covalent binding of molecules onto cellulosic nanomaterials is currently an intensive field of research. Functional linkers can be covalently grafted to the CNF surface through et al.20 Standard techniques  do not provide enough sensitivity and resolution to differentiate between low levels of adsorption and grafting or to understand reliably the surface chemistry of these key materials.20 Elemental analysis  and conductometric titration are often the only techniques available to indirectly probe surface modifications. In addition, and in line with a sustainable green strategy compatible with ecological concerns and medical applications, it is becoming increasingly important to perform these surface reactions in aqueous conditions employing readily available reagents/catalysts with minimal sensitivity to water. This further decreases the kinetic and activation efficiency of the carboxylic groups compared to the use of organic solvent.21 All in all, the lack of techniques able to report on low level of CNF surface modifications impedes the development of efficient, cost-effective and reproducible green synthetic routes and thus the widespread development of targeted and responsive drug-delivery CNF carriers.Nevertheless, access to unambiguous information regarding the surface chemistry of this type of system is currently lacking. This has recently been discussed in detail by Foster In this work, we show that dynamic nuclear polarization (DNP) enhanced solid-state NMR (ssNMR) can be used to unravel the surface chemistry of an innovative nanocellulose drug carrier even in the case of a very low level of grafting (<1 wt%), currently well beyond reach for all other techniques.22–24 Indeed, esterases are ubiquitous enzymes, available in fat tissues and at infection sites.25 This new CNF-t based complex represents thus a smart drug carrier formulation with “on-demand” API release abilities in the presence of esterases.More precisely we report on the implementation of a Diels–Alder reaction under heterogeneous aqueous conditions aiming at grafting the metronidazole drug onto CNF. CNFs-t were first amidated with furfurylamine in order to functionalize them with pending furan groups. Metronidazole was chemically modified with a maleimide derived carboxylic acid in order to introduce an ester function between the drug and the maleimide ring. The Diels–Alder reaction between the furan functionalized CNF-t (CNF-fur) and metronidazole containing maleimide, as depicted in et al. in 2011.7 The first step of functionalization corresponds to a catalyzed amidation with furfurylamine performed in water using a large excess of both reactants and reagents. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were used as coupling agents, and multiple washing steps were conducted in an effort to remove all the molecules that were not covalently bound, including the by-products issued from the coupling  and excess of amine. Metronidazole was esterified with the 6-maleimido-hexanoic acid in the presence of N,N′-dicyclohexylcarbodiimide (DCC) as coupling agent, to give the metronidazole-maleimide precursor that was then used in the next step  were employed with the aim to gather information regarding the surface chemistry  Magic Angle Spinning (MAS) NMR spectra of the different CNF samples are given in 13C CPMAS NMR spectra of the CNF-t sample.High-field dynamic nuclear polarization (DNP), an emerging hyperpolarization technique, is currently changing the scope of solid-state NMR spectroscopy since it allows enhancing the NMR sensitivity by several orders of magnitude.ents see . The DNP13C CPMAS spectrum of the TEMPO-oxidised CNF (CNF-t) shown in 23Na CPMAS spectra  between 63 ppm to 105 ppm, the carbonyl groups resulting from TEMPO-mediated oxidation pre-treatment of cellulose can be seen at 177 ppm in the DNP-enhanced 13C CPMAS NMR spectrum of CNF-fur displayed in etc.).42 It is thus not straightforward to differentiate between grafting and adsorption using the C7 resonance, especially since the peak intensities from the furan moieties are similar to the ones of the remaining coupling agents, suggesting a small amount of grafting.In the DNP-enhanced i.e. centrifugation–dispersion cycles and extensive dialysis, were employed to minimize the amount of remaining coupling agents. Nevertheless, DNP-enhanced NMR, as opposed to FTIR, UV and solid-state NMR, is still able to detect remaining traces.At this point, it is importing to mention that intense purification methods, vs. 175 ppm) results from a change in pH between the two samples, as described above. The two other resonances at 170 and 172 ppm are more challenging to assign. The chemical shift at 170 ppm (the less intense component) is consistent with an amide resonance resulting from the covalent bonding of furfurylamine with the surface carboxyl. The corresponding signal intensity of this component matches the intensity of the other furan resonances . Tentative assignment of the last component at 172 ppm is less obvious, with possible contributions from adsorbed NHS, as well as stable grafted by-products such as EDC-N-acylurea and/or NHS-ester  grafted on the CNF surface, as well as a potential contribution from adsorbed NHS.42 Resonances from aliphatic carbons of these reacted and unreacted coupling agents can be seen between 10–55 ppm, as shown in the inset of N-acylurea and EDC-urea, in agreement with previous studies.42,43 The relative intensity of furan (C8 and C11) and amide (at 170 ppm) resonances , such as N-acylurea from EDC and NHS-ester from NHS, but also to adsorbed NHS moieties.42,43Comparison of the relative integrals of C4 (or C1) and carbonyl resonances in CPMAS and MultiCP experiments of CNF-t allows the estimation of a CP-dynamics correction factor, which can then be used to further quantitatively analyse the DNP-enhanced 13C CPMAS NMR spectrum can be used to roughly estimate the degree of drug grafting to ∼1% compared to the glucose unit in cellulose. This is consistent with the amount of unreacted furfuryl in CNF-metro (estimated to ∼1% using the peak at 145 ppm) and the estimated yield of ∼50% for the Diels–Alder reaction step.Finally, the well-isolated metronidazole methyl resonance present in the CNF-metro DNP-enhanced †20,46 This can be used to confirm the presence of sulfur groups after acid hydrolysis46 and also to check surface modifications through the appearance of nitrogen or sulfur. Changes in proton, oxygen and carbon mass fractions are much more difficult to use because of the presence of contaminant and residual adsorbed water. As for conductometric titration, the degree of oxidation (DO) can be calculated for both unmodified and modified CNF samples. The difference in the DO value between the two samples is often used to estimate the amount of drug directly bound to CNF.20,21,26 Elemental analysis and conductometric titration were thus implemented on the three CNF samples  content.i.e. the presence of residual reduced TEMPO moieties as well as of β-alkoxy-elimination derivatives. Additionally, the presence of contaminants during the production process cannot be ruled out. From CNF-t to CNF-fur, the elemental analysis reports an increase in nitrogen and carbon mass proportion of ∼0.47% and ∼4%, respectively, while the oxygen wt% is decreased by ∼0.6%. These results are not consistent with the furfurylamine functionalization alone since an increase in nitrogen mass proportion of 0.47% should correspond to an increase of 1.9 and 0.5% in carbon and oxygen wt%, respectively. As a comparison, the DNP-enhanced NMR analysis estimates the amount of furfurylamine functionalization to be ∼2% with respect to glucose units in cellulose. This would translate into an increase of 0.2% and 0.9% in nitrogen and carbon wt%, respectively, which is much lower than the values observed by elemental analysis.Concerning the results of the elemental analysis on CNF-t (see Table S1N-acylurea and NHS ester) and remaining reduced TEMPO moieties. This has so far been overlooked in the interpretation of conductometric titration analysis.Conductometric titration estimates the number of glucose units bearing a carboxyl group in CNF-t to be ∼30.1%, while the DNP-enhanced NMR analysis gives ∼25%. This difference can partly be explained by the presence of remaining TEMPO moieties as well as other residual contaminants, which cannot be ruled out. Similarly to elemental analysis, conductometric titration overestimates the level of furfurylamine grafting as well. The analysis yields ∼12% of furan grafting as compared to ∼2% obtained by DNP-enhanced NMR. This difference can be largely explained by the presence of residual coupling agents bonded to CNF carboxylic acid groups (etc.), reduced TEMPO moieties, as well as the presence of β-alkoxy-elimination products. The presence of such organic moieties clearly impedes the reliable use of techniques such as elemental analysis and conductometric titration, which provide an overestimation of the drug loading without any means to differentiate between grafting and adsorption. This work should prove very useful to advance not only the field of cellulose-based smart drug carriers but also the full characterization of CNF-based materials.It has been shown that the large NMR sensitivity gains achieved by DNP enables the understanding of the surface chemistry of CNFs with unprecedented detail and the unambiguous characterization at natural isotopic abundance of low weight percentage of grafted API. Here, the API has on-demand release capability in the presence of esterases thanks to the design of a smart drug carrier formulation. The drug bonded to the CNF surface is estimated to about 1% of the glucose units of cellulose. Moreover, we show that DNP-enhanced NMR is the only technique that allows differentiating between grafting and adsorption in this case. Importantly, DNP-enhanced NMR spectroscopy also reveals the presence of other surface species, such as coupling agents , trifluoroacetic acid , metronidazole (CAS: 443-48-1), furfurylamine (CAS: 617-89-0), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride , N-hydroxysuccinimide , sodium hydroxide , hydrogen chloride  were purchased from Sigma Aldrich, Alfa Aesar or Acros Organics and used without further purification. Solution NMR spectra were recorded at room temperature in 5 mm tubes on a Bruker AC 400 MHz spectrometer . Chemical shifts (δ) are reported in parts per million (ppm) from low to high field and referenced to residual non-deuterated solvent relative to Me4Si. Standard abbreviations for multiplicity were used as follows: s = singlet; d = doublet; t = triplet; m = multiplet. High resolution mass spectrometry (HRMS) was carried out on a Waters Xevo G2-S-QTof mass spectrometer using ElectroSpray Ionisation .4-(Dimethylamino)pyridine , 47,48 , metronidazole  and DMAP  were dissolved in CH2Cl2 (25 ml) at 0 °C. DCC  was added after 15 minutes. The reacting mixture was stirred for 4 h at room temperature (rt), then filtered and concentrated under vacuum. The crude product was purified by flash chromatography on silica gel with CH2Cl2/MeOH 98/2 (v/v) as eluent to give the compound as a yellow amorphous solid . 1H NMR  δ 7.96, , 6.69 , 4.59 , 4.41 , 3.50 , 2.51 , 2.26 , 1.63–1.54 , 1.31–1.25 ; 13C NMR  δ 172.7, 170.8, 150.8, 134.1, 133.2, 62.4, 45.1, 37.5, 33.7, 28.2, 26.1, 24.1, 14.4; HRMS (ESI) m/z: calc. for C16H21N4O6 [M + H]+ 365.1461, obs. 365.1468. More details can be found in ESI Section 1.†6-Maleimido-hexanoic acidThe cellulose nanofibril (CNF) suspension was provided by the Centre Technique du Papier . Suspension of TEMPO-oxidized CNF (CNF-t) was produced from a pre-refined (40° SR) bleached bisulfite pulp provided by Rayonier Advanced Materials (previously TEMBEC). The pulp concentration was adjusted to 1.5 wt% and the oxidation was performed at pH 10 for 2 h in the presence of NaBr, NaClO, and the TEMPO reagent. A high pressure homogenizer from GEA  was used to defibrillate the oxidized cellulose fibers and produce the CNF-t suspension. The produced CNF-t suspension is in the form of a thick opaque gel at 1.6 wt%. AFM images of the low concentrated CNF-t suspension confirmed the nanosized morphology of CNF-t see . Using cThe CNF-t suspension concentration was decreased from 1.5 wt% to 0.4 wt% in order to be easily stirred. Deionized water was added before homogenization with an IKA Ultra-Turrax high shear mixer for 1 min at 10 000 rpm. The pH of the suspension was then adjusted to 4 under magnetic stirring using a 0.5 M HCl solution.A solution of the coupling agents EDC and NHS was prepared in deionized water and added to the suspension of CNF-t with a molar ratio of 4 equivalents of EDC and NHS for 1 equivalent of CNF-t surface carboxyl group (using the 30.1% DO observed by CT). The mixture was stirred for 30 min at rt at pH 4 and then the pH was increased to 8.5 before amine addition. Furfurylamine, with a molar ratio of 4 equivalents of amine for 1 equivalent of carboxylic acid of CNF-t, was mixed with 5 ml of deionized water and added to the mixture that was then stirred at rt with the pH maintained at 8.5.After 72 h of reaction, the reaction was quenched by decreasing the pH to 2–2.5 with 0.5 M HCl solution. To remove non-covalently bound chemicals , 6 cycles of washing, centrifugation (10 min at 11 100 rpm) and re-dispersion with a high shear mixer  for 1 min at rt were repeated. The last dispersion was done in neutral water to afford the furan modified CNF-t, referred to as CNF-fur.The last step of purification consisted in a dialysis of the CNF-fur suspension against neutral water with 6–8 kDa MWCO membranes  for at least 5 days under slow magnetic stirring and renewal of the medium twice a day.The Diels–Alder reaction was first optimized under homogeneous conditions before being implemented on the CNF-fur sample . The results obtained with AFM, conductometric titration, FTIR and elemental analysis can be found in ESI Sections 3 and 4.Characterization of the different CNF  were performed on thin films and/or cryogel powders. Thin films were produced by solvent casting in Petri dishes of 0.1 wt% CNF suspensions in order to obtain 30 g m30 in D2O. For CNF-t, 28 mg (aerogel) was impregnated with 40 μL of the DNP matrix (1). For CNF-fur and CNF-metro, 30 mg of each were impregnated with 80 μL of DNP matrix (1), an additional CNF sample was prepared following the synthesis used for CNF-fur, except that no furfurylamine was added. We refer to this sample as the CNF sample with coupling agents only. In this case, a DNP matrix (2) of 10 mM AsymPolPOK31 in glycerol/D2O/H2O  was used. 25 mg of CNF with coupling agents only was impregnated with 50 μL of DNP matrix (2). Each sample was then fully packed into a 3.2 mm outer-diameter sapphire rotor.DNP-enhanced NMR experiments were performed on the three CNF samples . The samples were prepared by impregnating the CNF powder with a DNP matrix (1) composed of 10 mM AMUPol1H resonance) DNP-NMR spectrometer equipped with 263 GHz gyrotron for microwave irradiation, a corrugated transmission line, and a low temperature 3.2 mm MAS probe used in double-resonance mode.49 All experiments were performed at a sample temperature of 100 K, using a magic-angle spinning (MAS) frequency of 13.3 kHz. Cross-polarization under MAS50 (CPMAS) experiments were performed with a radio frequency (rf) field strength of 50 kHz on the 13C while 1H rf field strengths were adjusted accordingly depending upon the ramp51 used to match CP conditions. The CP decay time constants were measured and were found to be in excess of 25 ms. The CP contact time was set to 2 ms for CPMAS experiments and 1 ms for the quantitative CP experiment using the Multi-CP45 pulse sequence. A 50-to-100% and 90-to-100% ramp were used for the 1H CP spin-lock in the CPMAS and quantitative CP using the MultiCP, respectively.51,52 The inter-scan delay was optimized according to the hyperpolarization build-up time of each sample, and set to 3.0 s for CNF-t, 2.5 s for CNF-fur, 1.9 s for CNF-metro and 1.9 s for CNF with coupling agents only. For the MultiCP experiment the inter-scan delay and the delay between each CP block were set to 3.9 s and 6 s, respectively. All experiments were processed and analyzed using Bruker Topspin 3.2 software, including the peak deconvolution and integration carried out to perform the analysis herein. 13C peak intensities are discussed relatively to carbon magnetization from the glucose unit of cellulose. The signal-to-noise of DNP-enhanced 13C CPMAS NMR spectra is about 500 : 1 for each of the carbon resonances of the glucose unit.All experiments were performed on a Bruker Avance III 400 MHz (FTIRFourier transform infra-redNMRNuclear magnetic resonanceDNPDynamic nuclear polarizationCNFsCellulose nanofibrilsThere are no conflicts to declare.SC-011-C9SC06312A-s001